Escherichia coli bl21 gold de3 cells

To prepare GST:Le-Dd10, Le-Dd10 cDNA fragments were prepared with the primers GSTLeDd10F and GSTLeDd10R (Table 1), digested with BamHI and EcoRI, and inserted into the BamHI and EcoRI site of pGEX-2 T, a GST fusion protein expression vector (GM Healthcare). After transforming into Escherichia coli BL21-Gold (DE3) cells (Agilent Technologies, CA, USA), transformants were incubated with 0.5 mM IPTG. GST:Le-Dd10 fusion proteins were adequately expressed at 37 °C for 16 h. The GST:Le-Dd10 was cut out from a 10% gel of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A 50-kDa target band was formed from the 26 kDa of GST and 24 kDa of Le-Dd10 protein. Gel slices from cells incubated at 37 °C for 16 h were stirred in 50 mM Tris–HCl buffer (pH 8.1) containing 0.1% SDS, 5% β -mercaptoethanol, 1 mM EDTA, and 0.2 mM phenylmethylsulfonyl fluoride at room temperature overnight. They were then suspended in PBS and subcutaneously and intraperitoneally injected into male rabbits to prepare the polyclonal antibody as previously described (Yoshida 1987 (link)). Four injections were performed at intervals of 10 days and rabbits were bled to death 1 week after the last injection. As control serum, sera were withdrawn from rabbits before the first injection.